The invention relates to an anti-HSV antibody as defined in the claims, a pharmaceutical composition comprising of an effective amount of the said antibody, an expression vector comprising of a nucleotide sequence which encodes the said antibody, a host cell comprising of the said nucleotide sequence, a hybridoma cell capable of producing the said antibody and the use of the said antibody as a drug, in particular to use for the manufacture of a drug for the prophylactical or therapeutical treatment of HSV-associated diseases in a subject; as defined in the claims.
The human pathogenic Herpes simplex virus (HSV) is a dermatotropic and neutrotropic DNA virus, whose clinical manifestations primarily originate from the skin and the nearby mucosa, and secondarily lead to neurological complications such as neuritis, meningitis, encephalitis, myelitis, polyradiculitis amongst others. At innate, acquired and also iatrogenic immune deficiency, to some extent severe progressions with a high lethality are reported. Due to the high infection rates of the population with HSV type 1 (HSV-1, 95%; H. labialis, H. cornea, eczema herpeticatum) and HSV type 2 (HSV-2, 10-30%; H. genitalis, H. neonatorum) and due to the often reactivation of the virus, latently persisting lifelong in the sensory and autonomic neural ganglions, HSV is of particular clinical relevance. Independent from the type of virus, the symptomatic therapeutic aims of primary or recidivating HSV infection are the inhibition of virus replication, shortening of the time of suffering, and prevention of the systemic complications influencing the frequency of recrudescence.
At early recognition and correct dosage, virustatic agents are successfully employed for antiviral therapy. The most common virustatic agents (e.g. acyclovir, penciclovir, foscarnet, idoxuridin) are nucleoside or pyrophosphate analogues, whose common active principle is based on the inhibition of DNA synthesis in virus-infected cells.
One of the most important therapeutic agents for the treatment of HSV infections is the purine nucleoside analogue acyclovir. It is phosphorylated by the viral thymidine kinase and then interferes with the viral DNA polymerase. In contrast, the human DNA polymerase is less susceptible against acyclovir by factor 30-50, for which reason merely marginal side effects are observed.
However, despite the development of selectively acting virustatic agents, chemotherapeutic treatment of viral diseases still represents a serious problem. In particular, the development of resistant strains against common chemotherapeutic agents observed during long-lasting prophylactic and therapeutic treatment of immunosuppressed patient is problematic. As a result, in more than 10% of the cases, due to the lack of effective virustatic agents, a fast progressing generalised infection with lethal progression is observed.
Presently, the pyrophosphate analogue foscarnet is particularly employed in immunosuppressed patients against acyclovir-resistant herpes virus. This agent causes a direct inhibition of the viral DNA polymerase and has no influence on the thymidine kinase. However, the use of foscarnet leads to severe undesirable side effects such as renal failure, cardiac problems, has toxicity on the bone marrow, and may also cause cutaneous ulceration. Because of its teratogenic effects, foscarnet may also not be administered during pregnancy. Further, the formation of cross-resistant strains is observed, which makes the development of alternative therapeutic agents highly necessary. A passive immunoprophylaxis is currently not available. A couple of experimental vaccine for active immunisation against HSV1 and HSV2 showed no verifiable success.
Antibodies hold great promise for the treatment of cancer, autoimmune disorders, and viral infections. JP 6135854 describes a therapeutic agent for herpes simplex virus infection in which a human monoclonal antibody against HSV and an antiviral nucleic acid analog such as acyclovir (ACV) are simultaneously or successively administered in the form of injection by intravenous drip infusion. DK 187286 discloses antibodies which exhibit multispecific immunoreactivities with regard to glycoprotein D (gD) from HSV-1 and HSV-2 (HSV gD-1 and gD-2). WO 1997/26329 describes human monoclonal antibodies useful for the diagnosis and treatment of HSV-1 and HSV-2. The latter antibody competes with the HSV 863 monoclonal antibody for binding to the glycoprotein D antigen of HSV-1 and HSV-2. U.S. Pat. No. 4,950,595 discloses a mouse-human hybridoma which produces an antivirus-human antibody, a process for preparation thereof, and an antivirus-human monoclonal antibody.
Furthermore, the humanization of another murine monoclonal antibody (Fd79) (Kogae et al., 1986) specific for HSV1/2 is described (Co, M. S. et al., 1991; Zeitlin L. et al., 1998). This antibody recognizes a shared epitope of glycoprotein B (gB) of HSV1 and HSV2. Moreover, humanized Fd79 has been produced in both transgenic plants and in the eukaryotic cell line SP2/0 and subsequently characterised, showing an affinity of 53 nM. Monoclonal murine antibody H1815 recognizes a similar but not identical epitope in the region of amino acids 263-283 of glycoprotein B (gB) (Navarro et al., 1992). However, H1815 is not capable of viral neutralization or inhibition of “cell-to-cell spread”. Finally, U.S. Pat. No. 6,180,370 describes humanized immunoglobulins and methods of making the same. Moreover, WO 2003/105782 pertains to specificity grafting of a murine antibody onto a human framework.
Thus, chemotherapeutic agents have undesired side effects and an increasing number of resistant strains is observed.
It is therefore an object of the invention to provide a (humanized) anti-HSV antibody which is capable of neutralising HSV infection and inhibiting cell-to-cell spread. Moreover, it is an object of the invention to provide a prophylactic and/or therapeutic agent for the treatment of HSV associated diseases which overcomes the above-mentioned disadvantages of conventionally applied chemotherapeutic agents.
Surprisingly, it was found that an antibody according to the invention solves this object. Accordingly, the present invention provides a promising alternative to the therapeutic agents known in the art for the treatment of HSV-infection on the basis of recombinantly produced antibodies, which may be humanized. These antibodies are capable of blocking both viral mechanisms to spread within a host. They effectively neutralize cell-free virus particles and inhibit the direct cell-to-cell spread of the virus. Since the antibodies specifically bind to a highly conserved epitope of the surface glycoprotein B (gB) of the HSV1 and HSV2 envelope, which is essential for the viral replication cycle, development of drug resistance is most unlikely.
Even though the effect of the murine antibody of the invention has in part already been described, c.f. Eis-Hübinger et al., Intervirology (1991); 32:351-360 and Eis-Hübinger et al., Journal of General Virology (1993); 74: 379-385, the antibody itself or the sequence of the complementarity determining regions (CDR) of the antibody of the invention as well as the epitope with which it binds to has never been published or made available to the public. In summary, the (humanized) antibody provides one or more of the following advantages:                The efficacy of the murine monoclonal antibody of the invention has already been proven (c.f. Eis-Hübinger et al., 1991; Eis-Hübinger et al., 1993). Moreover, the inventors show in the Examples section that the humanized antibody of the invention is also able to neutralize in vitro HSV1 and HSV2 viral infection and to inhibit viral spreading by inhibiting the “cell-to-cell spread” mechanism. In the context of infection progression in humans, the human immune system is not capable to generate antibody specificities for efficiently preventing HSV1/2 typical cell-to-cell spread.        The often and long-lasting prophylactic as well as therapeutic application of conservative chemotherapeutic agents such as acyclovir and foscarnet leads to an increase of the formation of resistant viral strains. This problem of resistance may be overcome by the (humanized) anti-HSV antibody described herein, administered alone or in combination with a virustatic agent such a acyclovir and/or foscarnet, since it relies on a different mechanism of action.        The antibody described herein specifically binds an epitope of the HSV gB protein. Development of HSV-resistance against the antibody of the invention is not to be expected, since mutations in the gB-protein lead to loss of viral infectivity.        Patients in which the systemic administration of conventional virustatic agents is contraindicated profit particularly from the (humanized) antibody described herein.        